US12370210B2 - Carbon monoxide as a treatment for neurodegenerative disease - Google Patents

Carbon monoxide as a treatment for neurodegenerative disease

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US12370210B2
US12370210B2 US17/616,110 US202017616110A US12370210B2 US 12370210 B2 US12370210 B2 US 12370210B2 US 202017616110 A US202017616110 A US 202017616110A US 12370210 B2 US12370210 B2 US 12370210B2
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Stephen Gomperts
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0053—Mouth and digestive tract, i.e. intraoral and peroral administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0087—Galenical forms not covered by A61K9/02 - A61K9/7023
    • A61K9/0095—Drinks; Beverages; Syrups; Compositions for reconstitution thereof, e.g. powders or tablets to be dispersed in a glass of water; Veterinary drenches
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/08—Solutions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A61P25/16—Anti-Parkinson drugs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia

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  • Described herein are methods for treating neurodegenerative diseases, e.g., Parkinson's disease (PD), with carbon monoxide (CO), e.g., orally administered CO.
  • PD Parkinson's disease
  • CO carbon monoxide
  • AD Alzheimer's disease
  • PD Parkinson's disease
  • Current treatment strategies for these progressive and fatal diseases are symptomatic and do not slow the disease course.
  • neuroprotective therapies that can impact the course of PD and AD, and prolong survival of neurons and other brain cells.
  • compositions comprising carbon monoxide for use in a method of treating a neurodegenerative disease in a subject, the method comprising orally administering a therapeutically effective amount of the carbon monoxide composition to a subject in need thereof.
  • the neurodegenerative disease is Parkinson's disease, Alzheimer's disease, dementia with Lewy bodies, multiple systems atrophy, progressive supranuclear palsy, corticobasal degeneration, or Pick's disease, frontotemporal dementia due to TDP-43, progranulin, C9ORF72, or Creutzfeldt-Jacob Disease.
  • the methods include orally administering a paste, gel, foam, emulsion, Newtonian liquid, or non-Newtonian liquid in which CO is dissolved, e.g., the oral composition is formulated as a paste, gel, foam, emulsion, Newtonian liquid, or non-Newtonian liquid in which CO is dissolved.
  • the CO is dissolved in a carrier comprising water and/or oil.
  • the methods include administering a therapeutically effective dose of HBI-002 to the subject.
  • the methods include providing a dose sufficient to achieve at least 3, 4, 5, 6, 7, 8, 9, or 10%, up to about 12, 13, 14, 15, or 20% Carboxyhemoglobin (COHb)/total hemoglobin.
  • COHb Carboxyhemoglobin
  • the methods include providing a dose sufficient to achieve 3-12% Carboxyhemoglobin (COHb)/total hemoglobin.
  • the methods include administering a dose of 0.2 ml/kg to 10 ml/kg body weight.
  • FIGS. 2 A-C Effect of oral HBI-002 in the aSyn model.
  • HBI-002 (10 ml/kg) or vehicle (10 ml/kg) dosed daily for 3 wks following injection of AAV-aSyn or AAV with no transgene. Animal sacrifice 3 wks after AAV.
  • A. HBI-002 reduced loss of substantia nigra pars compacta (SNpc) tyrosine hydroxylase-positive (TH+) neurons.
  • C Stereological TH+ neuron counts in the SNpc are preserved after HBI-002 treatment compared to Vehicle (p ⁇ 0.01, t-test). Analyses blind to treatment. Mean ⁇ std error.
  • HO-1 heme oxygenase 1
  • GDNF Growth Derived Neurotrophic Factor
  • FIGS. 7 A-C Effect of single dose inhaled CO in the MPTP model.
  • CO 225 ppm; 1 hr) or air (1 hr) administered 1 hr after MPTP or saline injection (normal control saline data not shown; no difference between groups in normal control animals).
  • B CO reduced loss of substantia nigra pars compacta (SNpc) tyrosine hydroxylase-positive (TH+) neurons.
  • C Stereological nigral TH+ neuron counts after CO treatment or air (P ⁇ 0.05, t-test). Mean ⁇ std error.
  • FIGS. 8 A-B HBI-002 treatment increased expression of HO-1 in hippocampus in the APP/PS1 mouse model of AD (A) and wild type mice (B).
  • inhaled low dose CO is associated with a certain amount of risk as well as adverse feeling in the art due to its association with toxicity.
  • CO at high doses is known to be neurotoxic and potentially fatal, causing irreversible brain damage and parkinsonism in survivors, including damage to critical brain structures such as the globus pallidus and the hippocampus.
  • exposure to gaseous CO was previously reported to be associated with an increased risk of dementia (Chang et al., PLoS ONE 9(8): e103078. doi:10.1371/journal.pone.0103078).
  • the present disclosure demonstrates that oral formulations that deliver CO are safe and therapeutically useful in PD and AD.
  • the disorders described herein include methods for the treatment of disorders associated with neurodegeneration.
  • the disorder is an alpha-synucleinopathy, e.g., PD, dementia with Lewy bodies, or multiple systems atrophy.
  • the disorder is a combined amyloidopathy and tauopathy, e.g., AD, or a tauopathy, e.g., progressive supranuclear palsy, corticobasal degeneration, or Pick's disease, or another proteinopathy, e.g., frontotemporal dementia due to TDP-43, progranulin, or C9ORF72, or a prionopathy, e.g., Creutzfeldt-Jacob Disease.
  • the methods include administering a therapeutically effective amount of an oral formulation of CO as described herein, e.g., HBI-002, to a subject who is in need of, or who has been determined to be in need of, such treatment.
  • the present methods can include administration of oral CO, e.g., in a liquid, including Newtonian and non-Newtonian liquids, such as pastes, gels, foams, emulsions, and other non-gaseous compositions, in which CO is dissolved at an amount that, when administered to a subject, provides a therapeutically or prophylactically effective amount of CO to the subject. See, e.g., U.S. Pat. No. 9,980,981.
  • the results presented herein demonstrate efficacy in models of PD and AD.
  • to “treat” means to ameliorate at least one symptom of the disorder associated with neurodegeneration, or to slow progression of the disorder; the present methods can also be used to reduce a subject's risk of developing the disease (e.g., in subjects who have REM sleep behavioral disorders or a family history or genetic predisposition to develop the disease).
  • Symptoms of Parkinson's disease include: tremors, slowness of voluntary movements, change in gait, and unsteady balance, in addition to nonmotor symptoms including cognitive dysfunction such as memory loss, confusion, loss of ability to plan or complete familiar tasks, withdrawal, hallucinations, and mood changes; thus, administration of a therapeutically effective amount of a compound described herein to a subject with PD can result in a reduction in any of these symptoms, a return or approach to normal motor control and nonmotor function, or the mitigation of further loss of function.
  • Symptoms of cognitive dysfunction associated with Alzheimer's disease include: memory loss, confusion, loss of ability to plan or complete familiar tasks, speech deficits, withdrawal, and mood changes; thus, administration of a therapeutically effective amount of a compound described herein to a subject with AD can result in a reduction in any of these symptoms, a return or approach to normal cognitive function, or the mitigation of further loss of function.
  • the method can result in increasing the life span of the subject.
  • the method can result in an improvement in the movement and/or motor function and/or cognitive function of the subject.
  • Administering may be performed, e.g., at least once (e.g., at least 2-times, at least 3-times, at least 4-times, at least 5-times, at least 6-times, at least 7-times, at least 8-times, at least 9-times, at least 10-times, at least 11-times, at least 12-times, at least 13-times, or at least 14-times) a week.
  • monthly treatments e.g. administering at least once per month for at least 1 month (e.g., at least two, three, four, five, or six or more months, e.g., 12 or more months), and yearly treatments (e.g., administration once a year for one or more years).
  • an oral formulation of CO is administered daily for subjects with PD and related disorders and for all patients at risk of developing PD and related disorders.
  • PD alpha-synuclein
  • DA dopamine
  • SN substantia nigra pars compacta
  • neuroinflammation appears to be an important contributor to PD pathogenesis 25-27 .
  • PD is a leading cause of death and long-term disability worldwide. In the US, ⁇ 500,000 Americans live with PD, with ⁇ 50,000 patients diagnosed annually 32 . The annual cost of PD in the US is approximately $25 billion 33 .
  • AD Alzheimer's disease
  • a cascade of neurodegeneration underlies the relentless, progressive, and irreversible cognitive failure characteristic of Alzheimer's disease (AD).
  • a ⁇ 1-42 aggregates in amyloid plaques, tau protein in neurofibrillary tangles and cell death comprise the classical neuropathological findings 34 .
  • Lewy bodies are frequently observed as well. 3536373839
  • recent evidence points to key roles for inflammation, mitochondrial dysfunction, Ca2+ dysregulation and aberrant neuronal activity 34,40 .
  • AD is a leading cause of death and disability and the most common form of dementia in the US, with ⁇ 5 million people living with AD in the US 41 .
  • the cost of AD to the US healthcare system is substantial, projected at approximately $200 billion in 2010 42 .
  • no neuroprotective agents have yet been identified to slow the course of this devastating and fatal disease, and finding such therapeutics remains a critical unmet need.
  • HBI-002 is a novel oral drug product containing CO that has been demonstrated to increase CO levels in animals in vivo without apparent safety issues ( FIG. 1 A ).
  • HBI-002 consists of a liquid formulation containing CO and generally recognized as safe (GRAS) substances (see U.S. Pat. No. 9,980,981).
  • GRAS safe
  • HBI-002 permits precise chronic delivery unlike other forms of CO, including iCO or carrier-molecule bound CO (CORMs).
  • Bioavailable CO with oral administration of HBI-002 has been demonstrated in mice ( FIG. 1 A ), documenting that oral HBI-002 administration leads to rapid CO bioavailability in the circulation measured as an increase in COHb saturation, with COHb level increasing in a dose-dependent manner.
  • the targeted dose of oral HBI-002 to achieve COHb levels of ⁇ 10% is achievable in rodents ( FIG. 1 A and Belcher et al. 44 ); this is well below the COHb level where toxicity is first observed ( ⁇ 20% 45 ; see below), and the targeted dose of oral HBI-002 to achieve COHb levels of 3-7% is achievable in subjects with PD and AD.
  • HBI-002 Since HBI-002 is given orally via a gavage needle, maximum doses are limited by stomach volume. A dose of 10 mL/kg is nearly a full stomach volume for a rat and therefore was chosen as our maximum once daily dose which leads to similar levels observed in smokers.
  • rats were randomly assigned to treatment groups. Experimenters were blinded to treatment group while performing stereotactic surgery to deliver viral particles and when preforming data collection and analysis. To ensure statistical power, experiments consisted of a minimum of 8 animals. In some cases, experimental endpoints were replicated in independent experiments. Data from these experiments are presented in aggregate in figures. The number of rats used in each experiment, replicated experiments, and the statistical test used are described in the figure legends.
  • HBI-002 was provided by Hillhurst Biopharmaceuticals, Inc. (Montrose, CA). HBI-002 was prepared following the procedure outlined in patent number U.S. Pat. No. 9,980,981. AAV vectors were purchased from Vigene Biosciences (Rockville, MD)
  • mice Female Sprague Dawley rats ranging from 220-240 g were purchased from Charles River (Wilmington, Mass.) and housed in Center for Comparative Medicine at Massachusetts General Hospital's Institute for Neurodegenerative diseases with a 12 hr light/dark cycle and access to food and water ad libitium. All experiments were approved by Massachusetts General Hospital's institutional animal care and use committee. After one week of acclimation, rats were anesthetized with isoflurane/oxygen and underwent bilateral stereotactic surgery as previously described with minor modifications.
  • Each animal received 2 ⁇ L of a 5 ⁇ 10 12 GC/mL viral titer injected into the nigra at the following coordinates: AP: ⁇ 5.2, ML: +/ ⁇ 2.0, DV: ⁇ 7.8.
  • the left nigra was given AAV1/2-CMV-empty vector-WPRE-BGH-polyA and the right nigra was given AAV1/2-CMV-human-A53T-alpha-sunuclein-WPRE-BGH-polyA. Animals were allowed to recover for 5 days before therapeutic interventions began.
  • HBI-002 or vehicle HBI-002 formulation without CO was given to animals via oral gavage (14 G needle) at a dose of 10 mL/kg/day. Both HBI-002 and vehicle were stored at 4° C. Freshly opened vials were stored on ice while in use and discarded within 5 minutes after opening to ensure consistent concentrations of CO.
  • DHBA 1,4 dihydroxybenzlamine hydrobromide
  • the supernatant was then filtered through Costar SpinX 0.22-micron spin filter cartridges. After filtering, 5 ⁇ L of supernatant was injected onto a Microsorb-MV column (C18, 150 mm ⁇ 5.6 mm, 5 micron) using an Ultimate 3000 UHPLC system (Thermo Fisher). Separation was achieved with a 17-minute isocratic method at a flow rate of 0.6 mL/min, and a mobile phase consisting of 75 mM sodium phosphate monobasic, 1.75 mM sodium-1-octanesulfonate, 100 ⁇ L/L triethylamine, 25 ⁇ M EDTA, and 10% acetonitrile.
  • Detection was carried out with an Ultimate 3000 ECD-3000RS (Thermo Fisher) with a screening electrode set to ⁇ 150 mV and a detection electrode set to 250 mV.
  • DHAB was used as a variable internal standard and dopamine was concentration was calculated from a standard curve.
  • Nuclear and cytosolic protein extracts were diluted in 2 ⁇ Laemmli sample buffer and heated at 70° C. for 10 minutes. After brief centrifugation, 20 ⁇ g of protein was loaded into each lane of 4-20% polyacrylamide gels. Electrophoresis was carried out at 150 V for 1 hour. Then, proteins were transferred to PVDF membranes, washed in distilled water, and dried overnight at 4° C. After reactivation of the PVDF membrane in methanol, total protein per lane was quantified using Revert 700 total protein stain and an Odyssey CLx imaging system (Licor, Lincoln, Nebr.) on the 700 nm channel per the manufacturer's instructions.
  • Revert 700 total protein stain and an Odyssey CLx imaging system (Licor, Lincoln, Nebr.) on the 700 nm channel per the manufacturer's instructions.
  • Membranes were then blocked in 5% dry non-fat milk powder in TBS for 2 hours at room temperature followed by overnight incubation at 4° C. in primary antibody diluted in 5% dry non-fat milk powder in TBS-T. After several washes in TBS-T, membranes were incubated in a 1:30,000 dilution of IRDye 800 donkey anti-rabbit (Licor, Lincoln, NE) in 5% dry non-fat milk powder and 0.02% SDS in TBS-T for 1 hour at room temperature. After several washes in TBS-T followed by a final wash in TBS, blots were imaged with an Odyssey CLx imaging system on the 800 nm channel. Fluorescent intensity of bands was determined using Image Studio (Licor, Lincoln, NE) and bands were normalized to total protein load in individual lanes.
  • Stereology was carried out as previously described with minor modifications. Sections labeled with TH, NeuN, and aSyn underwent counting using the optical fractionator principles with CAST stereology software (Olympus, Tokyo, Japan). Counting was limited to the substantia nigra and was done with a 20 ⁇ objective with a meander sampling of 100% to count the entire region. A total of eight sections were counted per animal.
  • Example 1 HBI-002 is Neuroprotective in PD
  • This Example explores the neuroprotective potential of CO for PD, providing data in two well established PD models: (i) rat AAV- ⁇ -synuclein (aSyn) genetic model and (ii) the mouse MPTP toxin model.

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Abstract

Methods for treating neurodegenerative diseases, e.g., Parkinson's disease (PD), with carbon monoxide (CO), e.g., orally administered CO.

Description

CLAIM OF PRIORITY
This application is a § 371 National Stage Application of PCT/US2020/036433, filed Jun. 5, 2020, which claims the benefit of U.S. Provisional Application Ser. No. 62/858,015, filed on Jun. 6, 2019. The entire contents of the foregoing are incorporated herein by reference.
TECHNICAL FIELD
Described herein are methods for treating neurodegenerative diseases, e.g., Parkinson's disease (PD), with carbon monoxide (CO), e.g., orally administered CO.
BACKGROUND
No treatments exist to reverse, arrest, or slow the course of neurodegenerative diseases including Alzheimer's disease (AD) and Parkinson's disease (PD), the most common neurodegenerative diseases worldwide and leading causes of death and long-term disability. Current treatment strategies for these progressive and fatal diseases are symptomatic and do not slow the disease course. Thus, there is a significant need to identify neuroprotective therapies that can impact the course of PD and AD, and prolong survival of neurons and other brain cells.
SUMMARY
In both the genetic AAV-αsynuclein A53T (AAV-aSyn) rat model and the toxin MPTP (MPTP) mouse model of PD, the present data show that CO exposure reduced subsequent loss of striatal dopamine and reduced subsequent loss of midbrain dopamine cells. Furthermore, CO exposure in the AAV-aSyn model reduced levels of aSyn, reduced numbers of aSyn aggregates, and reduced levels of toxic species of aSyn phosphorylated at serine 129. These results support the used of CO for neuroprotection in patients with PD and related disorders.
Thus provided herein are methods for treating a neurodegenerative disease in a subject, the method comprising orally administering a therapeutically effective amount of carbon monoxide to a subject in need thereof. Also provided are oral, e.g., liquid, compositions comprising carbon monoxide for use in a method of treating a neurodegenerative disease in a subject, the method comprising orally administering a therapeutically effective amount of the carbon monoxide composition to a subject in need thereof.
In some embodiments, the neurodegenerative disease is Parkinson's disease, Alzheimer's disease, dementia with Lewy bodies, multiple systems atrophy, progressive supranuclear palsy, corticobasal degeneration, or Pick's disease, frontotemporal dementia due to TDP-43, progranulin, C9ORF72, or Creutzfeldt-Jacob Disease.
In some embodiments, the methods include orally administering a paste, gel, foam, emulsion, Newtonian liquid, or non-Newtonian liquid in which CO is dissolved, e.g., the oral composition is formulated as a paste, gel, foam, emulsion, Newtonian liquid, or non-Newtonian liquid in which CO is dissolved.
In some embodiments, the CO is dissolved in a carrier comprising water and/or oil.
In some embodiments, the methods include administering a therapeutically effective dose of HBI-002 to the subject.
In some embodiments, the methods include providing a dose sufficient to achieve at least 3, 4, 5, 6, 7, 8, 9, or 10%, up to about 12, 13, 14, 15, or 20% Carboxyhemoglobin (COHb)/total hemoglobin.
In some embodiments, the methods include providing a dose sufficient to achieve 3-12% Carboxyhemoglobin (COHb)/total hemoglobin.
In some embodiments, the methods include administering a dose of 0.2 ml/kg to 10 ml/kg body weight.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described herein for use in the present invention; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.
Other features and advantages of the invention will be apparent from the following detailed description and figures, and from the claims.
DESCRIPTION OF DRAWINGS
FIG. 1A-C. Blood COHb levels in normal mice after three different doses of oral HBI-002. t=0 values are pre-treatment, n=3 mice for each time point of each dose.
FIGS. 2A-C. Effect of oral HBI-002 in the aSyn model. HBI-002 (10 ml/kg) or vehicle (10 ml/kg) dosed daily for 3 wks following injection of AAV-aSyn or AAV with no transgene. Animal sacrifice 3 wks after AAV. A. HBI-002 reduced loss of substantia nigra pars compacta (SNpc) tyrosine hydroxylase-positive (TH+) neurons. B. Compared to treatment with vehicle, treatment with HBI-002 preserved striatal DA (p=0.026, t-test; 10 vehicle and 8 HBI-002 rats/grp), C. Stereological TH+ neuron counts in the SNpc are preserved after HBI-002 treatment compared to Vehicle (p<0.01, t-test). Analyses blind to treatment. Mean±std error.
FIGS. 3A-B. Representative stereological photomicrographs and quantification of effect of HBI-002 treatment on NeuN+ cells in the substantia nigra. *** P=0.001, N=15
FIGS. 4A-B. Representative stereological photomicrographs and quantification of effect of HBI-002 treatment on aggregated aSyn in the substantia nigra. * P=0.0166, N=8
FIGS. 5A-B. Representative stereological photomicrographs and quantification of effect of HBI-002 treatment on phosphorylated serine 129 aSyn in the substantia nigra. ** P=0.0053, N=8.
FIGS. 6A-C. Quantification of effect of HBI-002 on brain levels of heme oxygenase 1 (HO-1) (P=0.038, N=14), Growth Derived Neurotrophic Factor (GDNF), and LC3B-II.
FIGS. 7A-C. Effect of single dose inhaled CO in the MPTP model. CO (225 ppm; 1 hr) or air (1 hr) administered 1 hr after MPTP or saline injection (normal control saline data not shown; no difference between groups in normal control animals). Animal sacrifice 5 days after MPTP. A. CO preserved striatal dopamine (DA) (P<0.03, t-test; 22 mice/grp). Striatal DA levels measured with HPLC, B. CO reduced loss of substantia nigra pars compacta (SNpc) tyrosine hydroxylase-positive (TH+) neurons. C. Stereological nigral TH+ neuron counts after CO treatment or air (P<0.05, t-test). Mean±std error.
FIGS. 8A-B. HBI-002 treatment increased expression of HO-1 in hippocampus in the APP/PS1 mouse model of AD (A) and wild type mice (B).
 DETAILED DESCRIPTION
No neuroprotective agents have yet been identified to prevent or slow the course of Parkinson's disease (PD) or Alzheimer's disease, progressive and fatal diseases, and finding such therapeutics is a research imperative.
Despite the known toxicity of CO at high concentration, CO at low concentration is now accepted as capable of regulating a host of physiologic processes1,2. Accumulating data have demonstrated that CO, at low concentrations, exerts key physiological functions in various models of tissue inflammation and oxidative injury. Specifically, the literature shows that low dose CO provides potent neuroprotection in PD model3. and in models of other neurological disease including including Alzheimer's4, traumatic brain injury5, stroke678-910, and multiple sclerosis11,12 as well as in in vitro models of apoptosis and oxidative injury13, 1314-1516. In each instance, the protection observed was associated with an inhibition in the inflammatory response or a reduction in oxidative stress and cell death. Importantly, low dose CO has been shown to upregulate Nrf2, a transcription factor associated with anti-inflammatory and anti-oxidative properties and implicated in PD1718-23. Moreover, CO readily crosses the blood-brain barrier, providing facile access to the CNS.
However, the use of inhaled low dose CO is associated with a certain amount of risk as well as adverse feeling in the art due to its association with toxicity. CO at high doses is known to be neurotoxic and potentially fatal, causing irreversible brain damage and parkinsonism in survivors, including damage to critical brain structures such as the globus pallidus and the hippocampus. Indeed, exposure to gaseous CO was previously reported to be associated with an increased risk of dementia (Chang et al., PLoS ONE 9(8): e103078. doi:10.1371/journal.pone.0103078).
Two formulations of CO have been studied to date in the clinic: (1) gaseous CO (iCO) provided by inhalation, and (2) CO bound to CO releasing molecules (CORMs) provided through intravenous injection. 25 phase I and II clinical trials have demonstrated safety of CO administration in the goal dose range of <10% CO-hemoglobin. However, considerations of dosing reliability, patient compliance, and patient and environmental safety complicate the use of CO gas, and toxicity of transition metal carriers, stability issues, and bioavailability issues have been found with CO administration via CORMs. These concerns are heightened with chronic administration, as would likely be needed in PD, AD, and other neurodegenerative diseases. Administration of CO via an oral formulation obviates these concerns, providing a safe and reliable dosing strategy for CO administration.
The present disclosure demonstrates that oral formulations that deliver CO are safe and therapeutically useful in PD and AD.
Methods of Treatment
The methods described herein include methods for the treatment of disorders associated with neurodegeneration. In some embodiments, the disorder is an alpha-synucleinopathy, e.g., PD, dementia with Lewy bodies, or multiple systems atrophy. In other embodiments, the disorder is a combined amyloidopathy and tauopathy, e.g., AD, or a tauopathy, e.g., progressive supranuclear palsy, corticobasal degeneration, or Pick's disease, or another proteinopathy, e.g., frontotemporal dementia due to TDP-43, progranulin, or C9ORF72, or a prionopathy, e.g., Creutzfeldt-Jacob Disease. Generally, the methods include administering a therapeutically effective amount of an oral formulation of CO as described herein, e.g., HBI-002, to a subject who is in need of, or who has been determined to be in need of, such treatment. Thus the present methods can include administration of oral CO, e.g., in a liquid, including Newtonian and non-Newtonian liquids, such as pastes, gels, foams, emulsions, and other non-gaseous compositions, in which CO is dissolved at an amount that, when administered to a subject, provides a therapeutically or prophylactically effective amount of CO to the subject. See, e.g., U.S. Pat. No. 9,980,981. The results presented herein demonstrate efficacy in models of PD and AD.
As used in this context, to “treat” means to ameliorate at least one symptom of the disorder associated with neurodegeneration, or to slow progression of the disorder; the present methods can also be used to reduce a subject's risk of developing the disease (e.g., in subjects who have REM sleep behavioral disorders or a family history or genetic predisposition to develop the disease). Symptoms of Parkinson's disease include: tremors, slowness of voluntary movements, change in gait, and unsteady balance, in addition to nonmotor symptoms including cognitive dysfunction such as memory loss, confusion, loss of ability to plan or complete familiar tasks, withdrawal, hallucinations, and mood changes; thus, administration of a therapeutically effective amount of a compound described herein to a subject with PD can result in a reduction in any of these symptoms, a return or approach to normal motor control and nonmotor function, or the mitigation of further loss of function. Symptoms of cognitive dysfunction associated with Alzheimer's disease include: memory loss, confusion, loss of ability to plan or complete familiar tasks, speech deficits, withdrawal, and mood changes; thus, administration of a therapeutically effective amount of a compound described herein to a subject with AD can result in a reduction in any of these symptoms, a return or approach to normal cognitive function, or the mitigation of further loss of function.
In some embodiments of the methods of treatment described herein, the method can result in increasing the life span of the subject.
In some embodiments of the methods of treatment described herein, the method can result in an improvement in the movement and/or motor function and/or cognitive function of the subject.
Administering may be performed, e.g., at least once (e.g., at least 2-times, at least 3-times, at least 4-times, at least 5-times, at least 6-times, at least 7-times, at least 8-times, at least 9-times, at least 10-times, at least 11-times, at least 12-times, at least 13-times, or at least 14-times) a week. Also contemplated are monthly treatments, e.g. administering at least once per month for at least 1 month (e.g., at least two, three, four, five, or six or more months, e.g., 12 or more months), and yearly treatments (e.g., administration once a year for one or more years).
In some embodiments, an oral formulation of CO is administered daily for subjects with PD and related disorders and for all patients at risk of developing PD and related disorders.
Parkinson's Disease (PD)
Although the neuropathology of PD is well-characterized, including aggregates of alpha-synuclein (aSyn) that accumulate in Lewy bodies in dopamine (DA) neurons of the substantia nigra pars compacta (SN) and other brain cells in association with their progressive degeneration, the etiology of PD is unclear. Oxidative stress, inflammation, and apoptosis all appear to be important contributors2425-2627. DA cells are particularly vulnerable to oxidative stress, as DA auto-oxidation produces reactive oxygen species that can impair mitochondrial function and induces apoptosis24,2829-3031. In addition, neuroinflammation appears to be an important contributor to PD pathogenesis25-27. PD is a leading cause of death and long-term disability worldwide. In the US, ˜500,000 Americans live with PD, with ˜50,000 patients diagnosed annually32. The annual cost of PD in the US is approximately $25 billion33.
It is well supported that smoking is linked to a protective effect in PD. However, the strong hypothesis in the scientific community has been that nicotine was the active neuroprotective molecule, in part because of the known neurotoxic effect of CO at high doses. However, two clinical studies with nicotine, the most rigorous of which only recently concluded, showed no protective effect from nicotine in PD patients.
Methods for identifying a subject with PD are known in the art.
Alzheimer's Disease
A cascade of neurodegeneration underlies the relentless, progressive, and irreversible cognitive failure characteristic of Alzheimer's disease (AD). Aβ1-42 aggregates in amyloid plaques, tau protein in neurofibrillary tangles and cell death comprise the classical neuropathological findings34. Lewy bodies are frequently observed as well.3536373839 In addition, recent evidence points to key roles for inflammation, mitochondrial dysfunction, Ca2+ dysregulation and aberrant neuronal activity34,40. AD is a leading cause of death and disability and the most common form of dementia in the US, with ˜5 million people living with AD in the US41. The cost of AD to the US healthcare system is substantial, projected at approximately $200 billion in 201042. To date, no neuroprotective agents have yet been identified to slow the course of this devastating and fatal disease, and finding such therapeutics remains a critical unmet need.
HO-1, the enzyme that produces endogenous CO among other catabolites, has been shown to be up-regulated in Alzheimer's disease, leading many in the field to hypothesize that the up-regulation of HO-1, such as occurs with CO, could be neuroprotective in Alzheimer's disease (AD). Moreover, preclinical in vivo studies have been conducted demonstrating that elevation of HO-1 is associated with improved cognitive function43 and neuroprotection41,42.
Methods for identifying a subject with AD are known in the art.
HBI-002
HBI-002 is a novel oral drug product containing CO that has been demonstrated to increase CO levels in animals in vivo without apparent safety issues (FIG. 1A). HBI-002 consists of a liquid formulation containing CO and generally recognized as safe (GRAS) substances (see U.S. Pat. No. 9,980,981). HBI-002 permits precise chronic delivery unlike other forms of CO, including iCO or carrier-molecule bound CO (CORMs). Bioavailable CO with oral administration of HBI-002 has been demonstrated in mice (FIG. 1A), documenting that oral HBI-002 administration leads to rapid CO bioavailability in the circulation measured as an increase in COHb saturation, with COHb level increasing in a dose-dependent manner.
The targeted dose of oral HBI-002 to achieve COHb levels of <10% is achievable in rodents (FIG. 1A and Belcher et al.44); this is well below the COHb level where toxicity is first observed (˜20%45; see below), and the targeted dose of oral HBI-002 to achieve COHb levels of 3-7% is achievable in subjects with PD and AD.
EXAMPLES
The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.
Material and Methods
The following materials and methods were used in Example 1 below.
Study Design
AAV-aSyn Experiments
Our objective was to investigate the ability of CO therapy to mitigate the cytotoxic effects of pathogenic aSyn in vivo. Our strategy was to use an established adeno-associated virus (AAV) expression system to overexpress mutant human aSyn in rats which leads to a pathology that closely mimics human PD pathology. Because the substantia nigra has limited connectivity across hemispheres, we performed bilateral stereotactic surgery to deliver viral particles directly into the substantia nigra. One hemisphere received AAV overexpressing aSyn while the other received the same expression system with an empty vector to act as an internal control. Following post-operative recovery, rats were treated with single daily doses of HBI-002 at a dose of 10 mL/kg. Since HBI-002 is given orally via a gavage needle, maximum doses are limited by stomach volume. A dose of 10 mL/kg is nearly a full stomach volume for a rat and therefore was chosen as our maximum once daily dose which leads to similar levels observed in smokers.
After 16 days of treatment with HBI-002, we performed quantitative analysis on the amount of dopamine present in the striatum using HPLC-ECD. We also used stereology to count the number of dopaminergic cells in the substantia nigra using tyrosine hydroxylase (TH) as a marker. Since the overexpression of proteins or the use of xenobiotics could potentially lead to changes in TH expression patterns, we also stained for the general neuronal marker NeuN and performed stereology to control for any potential variation on TH expression.
Since the aggregation and phosphorylation of aSyn are found in PD, we investigated the effect of CO therapy on these endpoints as well. We employed the use a monoclonal antibody specific to human aSyn to assess the extent of aSyn pathology. Similarly, we used an antibody specific to aSyn with a phosphorylated serine 129 residue.
We investigated the mechanism by which CO exerts a neuroprotective effect by first examining the multiple therapeutic targets described in related indications with CO intervention. These potential routes were investigated by assessing changes in protein expression by western blotting or by assaying increased nuclear factor activity with ELISAs.
In all experiments, rats were randomly assigned to treatment groups. Experimenters were blinded to treatment group while performing stereotactic surgery to deliver viral particles and when preforming data collection and analysis. To ensure statistical power, experiments consisted of a minimum of 8 animals. In some cases, experimental endpoints were replicated in independent experiments. Data from these experiments are presented in aggregate in figures. The number of rats used in each experiment, replicated experiments, and the statistical test used are described in the figure legends.
Reagents
HBI-002 was provided by Hillhurst Biopharmaceuticals, Inc. (Montrose, CA). HBI-002 was prepared following the procedure outlined in patent number U.S. Pat. No. 9,980,981. AAV vectors were purchased from Vigene Biosciences (Rockville, MD)
Overexpression of Human A53T aSyn in the SNpc of Rats
Female Sprague Dawley rats ranging from 220-240 g were purchased from Charles River (Wilmington, Mass.) and housed in Center for Comparative Medicine at Massachusetts General Hospital's Institute for Neurodegenerative diseases with a 12 hr light/dark cycle and access to food and water ad libitium. All experiments were approved by Massachusetts General Hospital's institutional animal care and use committee. After one week of acclimation, rats were anesthetized with isoflurane/oxygen and underwent bilateral stereotactic surgery as previously described with minor modifications. Each animal received 2 μL of a 5×1012 GC/mL viral titer injected into the nigra at the following coordinates: AP: −5.2, ML: +/−2.0, DV: −7.8. The left nigra was given AAV1/2-CMV-empty vector-WPRE-BGH-polyA and the right nigra was given AAV1/2-CMV-human-A53T-alpha-sunuclein-WPRE-BGH-polyA. Animals were allowed to recover for 5 days before therapeutic interventions began.
Low Dose CO Therapy
HBI-002 or vehicle (HBI-002 formulation without CO) was given to animals via oral gavage (14 G needle) at a dose of 10 mL/kg/day. Both HBI-002 and vehicle were stored at 4° C. Freshly opened vials were stored on ice while in use and discarded within 5 minutes after opening to ensure consistent concentrations of CO.
Quantification of Dopamine by HPLC-ECD
Dopamine was quantified as previously described with minor modifications. Rats were deeply anesthetized with ketamine and xylazine (dose) followed by rapid decapitation and brain removal. The striatum from each hemisphere was segregated, dissected on ice, and frozen on dry ice. Pieces of frozen striatum were weighed and homogenized in buffer containing 0.1 mM EDTA, 1 μM 3,4 dihydroxybenzlamine hydrobromide (DHBA, internal standard), and 50 mM phosphoric acid in a 1:20 ratio (weight: volume). The resulting homogenate was centrifuged at 14,000×g to pellet cell debris and precipitated protein. The supernatant was then filtered through Costar SpinX 0.22-micron spin filter cartridges. After filtering, 5 μL of supernatant was injected onto a Microsorb-MV column (C18, 150 mm×5.6 mm, 5 micron) using an Ultimate 3000 UHPLC system (Thermo Fisher). Separation was achieved with a 17-minute isocratic method at a flow rate of 0.6 mL/min, and a mobile phase consisting of 75 mM sodium phosphate monobasic, 1.75 mM sodium-1-octanesulfonate, 100 μL/L triethylamine, 25 μM EDTA, and 10% acetonitrile. Detection was carried out with an Ultimate 3000 ECD-3000RS (Thermo Fisher) with a screening electrode set to −150 mV and a detection electrode set to 250 mV. DHAB was used as a variable internal standard and dopamine was concentration was calculated from a standard curve.
Preparation of Nuclear and Cytosolic Extracts
Unfixed brain tissue from the substantia nigra was collected after 16 days of HBI-002 or vehicle treatment. Nuclear and cytosolic extracts were prepared using a nuclear extraction kit (Abcam, ab113474) following the manufacturer's instructions. After extraction, protein concentration was determined using a NanoDrop microvolume spectrophotometer (Thermo Fisher).
Immunohistochemistry
After 16 days of HBI-002 or vehicle treatment, animals were deeply anesthetized with ketamine and xylazine (dose) followed by rapid decapitation and brain removal. Brains were fixed for 3 days in 4% paraformaldehyde. After that time, brains were sectioned at 40 μm on a vibratome (Leica, Buffalo Grove, Ill.). Sections were collected in a 1 in 6 series and stored in PBS at 4° C.
For each antigen, a single series of sections were stained from each animal. Free-floating sections were incubated in 3% hydrogen peroxide for 15 minutes to block endogenous peroxidase. Next, sections were blocked and permeabilized in PBS with 2.5% bovine serum albumin (BSA), 10% normal goat serum (NGS), and 0.3% Triton X-100 for 30 minutes. Sections were then transferred to wells containing the corresponding primary antibody diluted in PBS with 2.5% BSA and 10% NGS. Sections were allowed to incubate overnight at 4° C. After several washes in PBS, antigens were visualized using an avidin-biotin detection system (ABC elite kit, Vector, Burlingame, Calif.) with ImmPact VIP and DAB substrates (Vector, Burlingame, Calif.) following the manufacturers' instructions. Sections were mounted, dehydrated in graded ethanol, cleared in xylene, and cover slipped with permanent mounting media (VectaMount, Vector, Burlingame, Calif.). Staining intensity was quantified as previously reported.
Immunoblotting
Nuclear and cytosolic protein extracts were diluted in 2× Laemmli sample buffer and heated at 70° C. for 10 minutes. After brief centrifugation, 20 μg of protein was loaded into each lane of 4-20% polyacrylamide gels. Electrophoresis was carried out at 150 V for 1 hour. Then, proteins were transferred to PVDF membranes, washed in distilled water, and dried overnight at 4° C. After reactivation of the PVDF membrane in methanol, total protein per lane was quantified using Revert 700 total protein stain and an Odyssey CLx imaging system (Licor, Lincoln, Nebr.) on the 700 nm channel per the manufacturer's instructions. Membranes were then blocked in 5% dry non-fat milk powder in TBS for 2 hours at room temperature followed by overnight incubation at 4° C. in primary antibody diluted in 5% dry non-fat milk powder in TBS-T. After several washes in TBS-T, membranes were incubated in a 1:30,000 dilution of IRDye 800 donkey anti-rabbit (Licor, Lincoln, NE) in 5% dry non-fat milk powder and 0.02% SDS in TBS-T for 1 hour at room temperature. After several washes in TBS-T followed by a final wash in TBS, blots were imaged with an Odyssey CLx imaging system on the 800 nm channel. Fluorescent intensity of bands was determined using Image Studio (Licor, Lincoln, NE) and bands were normalized to total protein load in individual lanes.
Stereology
Stereology was carried out as previously described with minor modifications. Sections labeled with TH, NeuN, and aSyn underwent counting using the optical fractionator principles with CAST stereology software (Olympus, Tokyo, Japan). Counting was limited to the substantia nigra and was done with a 20× objective with a meander sampling of 100% to count the entire region. A total of eight sections were counted per animal.
Statistical Analysis
All data was analyzed using Prism 8 (GraphPad) with unpaired, two-tailed t-tests. In all experiments, alpha was predetermined to be set at 0.05. Effect sizes and experimental power were calculated using G*Power 3.1 (University of Dusseldorf, Dusseldorf, Germany).
Example 1. HBI-002 is Neuroprotective in PD
This Example explores the neuroprotective potential of CO for PD, providing data in two well established PD models: (i) rat AAV-α-synuclein (aSyn) genetic model and (ii) the mouse MPTP toxin model.
In the AAV-aSyn rat model, AAV is used to deliver human aSyn harboring the A53T mutation directly into the SNpc, leading to cell loss over 3 weeks in association with aSyn oligomerization46,47 (FIG. 2A).48,49,50. After AAV-aSyn was injected into the right SNpc and AAV containing no aSyn transgene was injected into the left SNpc, rats were treated with either HBI-002 (10 mL/kg; n=8) or vehicle (10 mL/kg; n=10) via oral gavage, daily for 3 wks. Striata and midbrains were harvested, and we measured DA levels in the left and right striata via HPLC, blinded to treatment. Rats treated with vehicle showed preservation of only 36.05%±6.56% of right striatal DA compared to the left side. In contrast, rats treated with HBI-002 retained 74.09%±11.85% of right striatal DA compared to left (p=0.026, t-test) (FIG. 2B). We next used standard stereological methods to count tyrosine hydroxylase (TH)-positive neurons in the SNpc, blinded to treatment. Critically, while vehicle was associated with reduction of right SNpc TH+ neurons to 36.2%±1.82% compared to the left side, HBI-002 treatment was associated with increased preservation of right-sided TH+ cell counts to 75.7%±3.73% compared to the left (p=0.0001, t-test) (FIG. 2C). The same treatment also reduced loss of nigral cells (p=0.0001, t-test) (FIGS. 3A-B).
In addition, treatment with HBI-002 reduced synuclein aggregates (p=0.0166, t-test) (FIG. 4A-B). HBI-002 treatment also reduced levels of synuclein phosphorylated on Ser129 (p=0.0053, t-test) (FIGS. 5A-B), a pathological feature of PD that potentially contributes to aSyn A53T toxicity51.
Biochemical analysis showed engagement of HO-1 (p=0.038, t-test), GDNF, and LC3B-II (FIGS. 6A-C).
These results indicate that oral CO is neuroprotective in an animal model of PD.
We also employed a short-term regimen of the widely used5253 MPTP toxin model in which DA loss ensues within 5-8 days of MPTP. We elected to initiate iCO treatment only after MPTP exposure in order to model protective treatment of human PD, which begins only after disease onset. We exposed mice to MPTP (40 mg/kg, i.p.)54,55 or saline (study control) and then treated with a single dose of either iCO (225 ppm) or air (negative control) for 1 hr, for 4 total groups: 1) MPTP/CO (225 ppm; 1 hr), 2) MPTP/air (1 hr), 3) saline/CO (225 ppm; 1 hr), 4) saline/air (1 hr). Five days after MPTP exposure, we measured DA levels in the striata via HPLC and counted TH+ neurons in all groups. MPTP mice treated with CO had ˜46% higher DA levels than those treated with air (2-tailed t-test, p=0.028; FIG. 7A). CO did not alter DA levels in saline-treated mice (air: 96.7±1.9 pmol/mg, iCO: 97.0±2.4 pmol/mg, p>0.05, n=22 per group). Critically, for TH+ neurons, MPTP mice treated with iCO had ˜22% more TH+ neurons than those treated with air (p<0.05; FIGS. 7B-C). In contrast, TH+ cell counts in saline-treated mice were not significantly different: air 7,536±541; iCO 7,437±545, p>0.05. These results in both a genetic model of PD and a toxin model of PD strongly support therapeutic efficacy of HBI-002 for PD.
Example 2. HBI-002 is Neuroprotective in AD
Endogenous CO is generated in the body and brain by the heme oxygenases (HO). These enzymes degrade heme, a toxic species present in several mitochondrial proteins and hemoglobin, into CO, the antioxidant biliverdin, and Fe2+56. HO-1 is expressed in most cells of the body and brain57, and both neuronal and non-neuronal CNS cells, including astrocytes and microglia, rapidly upregulate HO-1 in response to stress58. HO-1 expression is significantly elevated in the AD brain, with 9-fold elevation in the hippocampus relative to normal controls59. Furthermore, in AD, HO-1 colocalizes with tau pathology, including the neurites of senile amyloid plaques, neurofibrillary tangles, and neuropil threads60. Potentially due to the divergent properties of HO-1's enzymatic products, including the neuroprotective effects of CO outlined above, the anti-oxidant properties of biliverdin (which is modified to the anti-oxidant bilirubin), and the potential pro-oxidant effects of iron (Fe2+), which can deposit in the brain and has been linked to the progression of AD61, conflicting reports exist about the properties of HO-1 in AD, although several studies have found HO-1 to be protective in AD models62,63.
To determine whether oral formulation CO (HBI-002) can upregulate HO-1 to engage HO-1 cascades in an animal model of AD beta-amyloidopathy, we studied the effects of HBI-002 in 10-11 month old mice harboring mutations in the amyloid precursor protein (APP) and the presenilin (PSEN) protein (APPswe/PSEN1dE9), known as APP/PS1 mice6465). This AD mouse model forms robust amyloid plaques by 6 months.
APP/PS1 mice were treated with HBI-002 or Vehicle at a dose of 10 mL/kg, once daily, for 14 days. Mice ranged from 10-11 months in age. HO-1 levels in the hippocampus were determined with Western blot.
In APP/PS1mice, CO treatment via oral gavage was associated with increased HO-1 expression in the hippocampus (FIG. 8A). These results are consistent with findings in wild-type (WT) mice, where oral HBI-002 treatment also increased HO-1 expression (p=0.038, t-test; n=14 per group (FIG. 8B)).
These results show that oral CO formulation (HBI-002) increases HO-1 expression in the APP/PS1 model of AD. Together with prior results demonstrating that CO is neuroprotective in AD models66 and prior results linking HO-1 associated molecular cascades to neuroprotection in AD41,42 and to improved cognitive function26 these findings support therapeutic efficacy of an oral formulation CO in AD.
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Other Embodiments
It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims (8)

What is claimed is:
1. A method of treating a neurodegenerative disease in a subject, the method comprising orally administering a therapeutically effective amount of carbon monoxide (CO) to a subject in need thereof, wherein the therapeutically effective amount comprises a dose sufficient to achieve at least 3% and up to about 20% Carboxyhemoglobin (COHb)/total hemoglobin.
2. The method of claim 1, wherein the neurodegenerative disease is Parkinson's disease, Alzheimer's disease, dementia with Lewy bodies, multiple systems atrophy, progressive supranuclear palsy, corticobasal degeneration, or Pick's disease, frontotemporal dementia due to TDP-43, progranulin, C9ORF72, or Creutzfeldt-Jacob Disease.
3. The method of claim 1, comprising orally administering a paste, gel, foam, emulsion, Newtonian liquid, or non-Newtonian liquid in which CO is dissolved.
4. The method of claim 3, wherein the CO is dissolved in a carrier comprising water and/or oil.
5. The method of claim 1, comprising administering a therapeutically effective dose of HBI-002 to the subject.
6. The method of claim 5, comprising administering a dose of 0.2 ml/kg to 10 ml/kg body weight.
7. The method of claim 1, comprising providing a dose sufficient to achieve at least 4, 5, 6, 7, 8, 9, or 10%, up to about 12, 13, 14, 15, or 20% Carboxyhemoglobin (COHb)/total hemoglobin.
8. The method of claim 7, comprising providing a dose sufficient to achieve 3-12% Carboxyhemoglobin (COHb)/total hemoglobin.
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